PROJECT SUMMARY In this Project, we will leverage our novel Native Flexibly Linked (NFL) platform to generate super stable, soluble and well-ordered South African clade C 1086 trimers for eventual particulate and membrane- associated display based on our recently developed design strategies using other subtype C and B-derived envelope glycoproteins (Env) to generate ?super stable? trimers. We present such design improvement in the Preliminary Data of Aim 1. Alternatively, we will use other South African clade C Envs (i.e. CAP244) if transplantation of these modifications to 1086 proves difficult. The subtype A, BG505-derived Env, forms highly homogeneous, well-ordered native-like trimers by either the NFL or SOSIP design strategies. But since most HIV infected individuals are located in sub-Saharan Africa, there is an unmet need to generate stable spike mimetics of South African or other African origins as a starting point towards a vaccine designed specifically for the major focus of the HIV pandemic. The NFL trimers derived from other HIV-1 Env sequences, such as JRFL and 16055 (subtypes B and C, respectively) form NFL trimers to a lesser degree, requiring negative selection to generate homogenous, well-ordered spike mimetics. These differences in trimer propensity formation presumably emanate either from primary amino acid differences or post translationally related aspects, such as distribution of N-glycans on the Env solvent-exposed surfaces. To improve the ?spontaneous? trimer formation of NFLs derived from the clade B JRFL and C 16055 Envs, we performed both sequence and structural alignments to identify BG505 trimer-derived (TD) sequence-specific residues that might increase the propensity of the JRFL- and 16055-derived Env to more efficiently form well-ordered trimers. By this process, having identified key residues at the gp120-gp41 interface, as well as in the variable regions, we further stabilized these trimers by engineering an intra-protomer disulfide linkage in the pre-bridging sheet (201-433). This disulfide locks the gp120 in the pre-fusion state and prevents CD4 induced-conformational rearrangements. Both the TD mutations and the 201-433 disulfide linkage increased well-ordered trimer formation and thermostability. Here, we will expand this approach to 1086 (Aim 1) and then in Aim 2 we will generate cysteine-linked Env liposomes to create virus-like particles (VLPs). This strategy may overcome the low levels of Env incorporation that occurs using natural HIV Env and Gag sequences to generate VLPs. We will design and develop liposomal nanoparticles displaying high-density HIV Env NFL clade C (1086) spike mimetics, covalently link the trimers to the liposomes and analyze them by binding kinetics and negative staining followed by cryoEM. We will then cross-compare them to other means of particulate or membrane display described in other components of this P01. We will also generate the trimers for bulk or single B cell sorting and point mutant variants in Aims 3 and 4. The merging of well-ordered clade C trimers and particulate display represent a promising lead toward the development of a more effective HIV vaccine.